Five-Helix protein

ABSTRACT

Five-Helix protein, which comprises the three N-helices and at least two, but not three, of the three C-helices of the trimer-of-hairpin structure of HIV gp41, separated by linkers, such as amino acid residue linkers, is disclosed. Six-Helix protein, which includes the three N-helices and the three C-helices of the trimer-of-hairpin structure of HIV gp41, separated by linkers, is also disclosed.

RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisionalapplication No. 60/171,042, entitled “Five-Helix Protein,” by Michael J.Root, Michael S. Kay, David C. Chan and Peter S. Kim (filed Dec. 16,1999) and U.S. Provisional application No. 60/234,572, entitled “ProteinDesign of an HIV Entry Inhibitor,” by Michael J. Root, Michael S. Kay,David C. Chan and Peter S. Kim (filed Sep. 22, 2000). The entireteachings of both of the referenced provisional applications areincorporated herein by reference.

GOVERNMENT SUPPORT

[0002] The invention was supported, in whole or in part, by Grant NumberPO1 GM 56552 from National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] HIV is the virus that is responsible for the worldwide AIDSepidemic. The initial stages of HIV infection involve the fusion of theviral membrane with the target cell membrane, a process that injects theviral contents into the cellular cytoplasm. On the viral side, themolecular complex responsible for the fusion activity contains thesurface protein gp 120 and the transmembrane protein gp41. It iscurrently believed that gp120 interacts with the proteins CD4 andcoreceptors on the target cell, resulting in a conformational changethat causes gp41 to insert its amino terminus (fusion peptide region)into the target cell membrane. This structural rearrangement promotesthe fusion of virus and cellular membranes through a poorly understoodmechanism.

SUMMARY OF THE INVENTION

[0004] The present invention relates to a novel protein, referred to asFive (5)-Helix or Five-Helix protein, that, under the conditionsdescribed herein, folds into a stable structure, binds a peptide(referred to as C34) that corresponds to the C-peptide region of HIVgp41 protein or a portion of the region and inhibits HIV infection ofmammalian cells, such as human cells. Five-Helix is made up of the threeN-helices and at least two, but not three, of the three C-helices of thetrimer of hairpin structure of HIV gp41, separated by linkers, such asamino acid residue linkers. That is, Five-Helix includes the threeN-helices and at least two of the three C-helices of HIV gp41. It canalso include a portion of the third C-helix, but does not include theentire third C-helix. In each case, the helices are separated bylinkers, preferably amino acid residue linkers, between the precedingand following helices. In one embodiment, Five-Helix can be representedas: N-linker-C-linker-N-linker-C-linker-N, wherein N represents anN-helix and C represents a C-helix or C-helix portion. As used herein,the term Five-Helix or Five-Helix protein encompasses all suchembodiments (those including three N-helices and two or more, but lessthan three complete C-helices, separated by appropriate linkers). Theamino acid composition of Five-Helix can vary greatly, provided thatFive-Helix presents a surface that is structurally complementary to theC-peptide region of HIV gp41 protein and, preferably, binds C34 or theC-peptide region of gp41, as peptides or part of gp41 as a whole. Thatis, the remaining (interacting) surface of Five-Helix (the C-peptidebinding site, all or a portion of which is not occupied by a C-peptide)must be presented in such a manner (conformation) that it is availableto bind the C-peptide region of HIV gp41. In the case of vaccine andtherapeutic applications of Five-Helix, Five-Helix must bind (be capableof binding) C34 or the C-peptide region of HIV gp41. In the cases inwhich Five-Helix is used as a drug-screening tool or anantibody-screening tool, Five-Helix need not bind (need not be capableof binding) C34 or the C peptide region of HIV gp41.

[0005] In one embodiment, Five-Helix has the amino acid sequence of SEQID NO.: 1. In other embodiments, Five-Helix presents a surface that isstructurally complementary to the C-peptide region, preferably binds C34or the C-peptide region and has an amino acid sequence that differs fromthat of SEQ ID NO.: 1 by addition, deletion, substitution or alterationof at least one amino acid residue. The order of the N-helices andChelices of Five-Helix can also vary, provided that the conformation issuch that the exposed protein presents a surface structurallycomplementary to the C-peptide region of HIV gp41. The linkers can be ofany length or composition, provided that the Five-Helix proteinconformation, described above, is retained. Five-Helix can be an L-aminoacid protein, a D-amino acid protein or a combination of L-amino acidresidues and D-amino acid residues; these residues can be modifiedresidues.

[0006] The present invention further relates to DNA encoding Five-Helix;methods of producing Five-Helix; methods in which Five-Helix is used,such as in methods of inhibiting entry of HIV into mammalian cells,including human cells, and methods of eliciting an immune response in anindividual, such as a human; methods in which DNA encoding Five-Helix isused, such as in gene therapy methods; genetically engineered cells,such as bacteria, human and other mammalian and other eukaryotic cells,which contain and express Five-Helix protein-encoding DNA and methods ofusing such cells (e.g., for gene therapy or Five-Helix production);compositions, such as pharmaceutical compositions, which includeFive-Helix; Five-Helix complex comprising Five-Helix and a componentthat binds HIV envelope protein (e.g., gp120); compositions, such aspharmaceutical compositions, which include Five-Helix complex;antibodies, particularly neutralizing antibodies which bind Five-Helixand methods in which such antibodies are used, such as methods ofreducing-HIV infection; and methods of identifying molecules orcompounds that inhibit HIV infection of cells and/or bind the Five-Helixprotein.

[0007] Five-Helix is useful as an anti-HIV therapeutic agent, aprophylactic agent or drug to prevent HIV infection, a reagent foridentifying (screening for) or designing other anti-HIV therapeutics orprophylactics, and an immunogen to elicit antibodies that prevent orreduce HIV infection. In a specific embodiment, the invention relates toa method of identifying a compound or molecule that binds Five-Helix andinhibits HIV infection of mammalian cells, wherein the compound ormolecule to be assessed is referred to as a candidate inhibitor,comprising combining a candidate inhibitor and Five-Helix, underconditions appropriate for binding of an inhibitor and Five-Helix tooccur and determining if binding occurs, wherein if binding occurs, thecandidate inhibitor is a compound or molecule that binds Five-Helix. Themethod optionally further comprises determining if the compound ormolecule that binds Five-Helix inhibits HIV infection of mammalian(e.g., human) cells, such as in a cell-based assay. Such a compound ormolecule will inhibit (totally or partially) HIV infection of cells(e.g., by preventing or interfering with formation of thetrimer-of-hairpins).

[0008] In another embodiment, the invention relates to a method ofeliciting an immune response to HIV in an individual, comprisingintroducing, by an appropriate route, a composition comprisingFive-Helix and a physiologically acceptable carrier, in a dosesufficient to elicit the immune response in the individual. Vaccinescomprising Five-Helix (or a variant or portion thereof) in aphysiologically acceptable carrier are the subject of this invention.

[0009] Also the subject of the present invention is Six (6)-Helixprotein, which comprises three N-helices and three C-helices of HIVgp41, joined by linkers, such as amino acid residue linkers. In oneembodiment, Six-Helix protein comprises the amino acid sequence of SEQID NO.: 2. In other embodiments, the amino acid sequence of Six-Helixdiffers from that of SEQ ID NO.: 2 by addition, deletion, substitutionor alteration of at least one amino acid residue. Six-Helix protein isuseful not only for producing Five-Helix, but also as a negative controlin screening for drugs that inhibit membrane fusion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The file of this patent contains at least one drawing in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

[0011]FIGS. 1A and 1B illustrate targeting HIV-1 membrane fusion. FIG.1A is a schematic of HIV-1 membrane fusion depicting events that promoteformation of the gp41 trimer-of-hairpins. The N-terminal fusion peptideof gp41 (red), inaccessible in the native state, inserts into targetcell membranes following gp120 interaction with CD4 and coreceptors (notshown). Formation of the prehairpin intermediate exposes the N-terminalcoiled coil (gray), the target of C-peptide inhibition. This transientstructure collapses into the trimer-of-hairpins state that brings themembranes into close apposition for fusion. FIG. 1B shows the design ofthe 5-Helix construct. The ribbon diagrams (top) depict the corestructure of the trimer-of-hairpins (left and D. C. Chan et al., Cell89, 263-273 (1997)) and a model of 5-Helix (right). The inner grayhelices represent N36 peptides, and the outer blue helices represent C34peptides. One C-peptide has been removed in the model of 5-Helix andorange lines have been drawn to represent connectivity between thehelices. In the design of 5-Helix, the N40 and C38 sequences (given insingle-letter amino acid code) are alternately linked by short Gly/Serpeptide sequences (gray bars in schematic at bottom (See Example 1).

[0012] FIGS. 2A-2D show properties of 5-Helix. FIG. 2A is the circulardichroism (CD) apectrum of 5-Helix (10 μM) at 25° C. The spectrumindicates that the 5-Helix protein adopts >95% of the helical contentexpected from the design. FIG. 2B is a graphic representation of thermaldenaturation of 5-Helix monitored by ellipticity at 222 nm in TBS(filled squares) and in 3.7 M guanidine (Gu)HCl/TBS (open squares). Thedenaturation observed in the GuHCl solution is >90% reversible. FIG. 2Cshows results of nickel (Ni)-NTA precipitation of 5-Helix with aHis-tagged C-peptide. Untagged 5-Helix and His-tagged C-peptide (denotedC37-H6) were mixed before Ni-NTA agarose was added in order toprecipitate complexes containing C37-H6 (lanes 1 and 5 and Example 4).Addition of excess untagged C-peptide (C34) shifts the 5-Helix moleculesfrom the bound to the unbound fraction (lanes 2 and 6). FIG. 2D is theCD spectra of 5-Helix and C37-II6 before (filled squares) and after(open circles) mixing in a mixing cuvette. The increase in ellipticityat 222 nm upon mixing indicates an interaction between the two speciesthat increases the total helical content (corresponding to an additional28 helical residues per associated C-peptide).

[0013] FIGS. 3A-3C show results of assessment of 5-Helix inhibition ofHIV-1 envelope-mediated membrane fusion, as described in Example 2. FIG.3A shows results of assessment of titration of viral infectivity by5-Helix (filled squares), 6-Helix (open triangles), and 5-Helix(D4)(open circles), as described in Example 3 and 5 The data represent themean ±SEM of two or more separate experiments. FIG. 3B is a graphicrepresentation of antagonistic inhibitory activities of 5-Helix and C34.The number of syncytia were measured in a cell-cell fusion assayperformed in the absence or presence of 5-Helix, C34, or mixtures of5-Helix and C34 at the indicated concentrations. The IC₅₀ values for5-Helix and C34 in this assay are 13±3 nM and 0.55±0.03 nM, respectively(D. C. Chan et al., Proc. Natl. Acad. Sci. USA95, 15613-15617 (1998)).Data represent the mean and range of mean of duplicate measurements,except for the control (mean±SEM of five measurements). FIG. 3C showsresults of assessment of 5-Helix inhibition of pseudotyped viruscontaining different HIV-1 envelope glycoproteins. The reported IC₅₀values represent the mean±SEM of three independent experiments.

[0014]FIG. 4 is a helical wheel diagram depicting the interaction of5-Helix with the C-peptide region of gp41. The a through g positions ineach helix represent sequential positions in the 4,3-hydrophobic heptadrepeat in each sequence. The a and d positions in the gp41 C-peptideregion interact with the exposed e and g positions on the N40 coiledcoil of 5-Helix. Residues are boxed according to their degree ofconservation as determined from the alignment of 247 sequences fromHIV-1, HIV-2, and SIV isolates (HIV-1 sequence database, August 2000,Los Alamos National Laboratory): black rectangle, >90% identical; greyrectangle, >90% conservative substitution; dotted rectangle, 70-90%conserved; no box, <70% conserved. In generating FIG. 4, substitutionswithin the following groups of amino acid residues were considered to beconservative: [Asp, Glu], [Lys, Arg], [Asn, Gln], [Phe, Tyr], [Ser, Thr]and [Val, Ile, Leu, Met]. Note the high degree of conservation in the aand d positions of the C-peptide region of gp41, a property markedlylacking in other positions (particularly c and g) of the C-peptideregion not directly involved in binding 5-Helix.

[0015]FIG. 5 is the structural arrangement of HIV gp41. Helical regions(heptad repeats) are shown in grey, and the relative position of N-(N36)and C-(C34, DP178) peptides are indicated. In the ribbon diagram of thehelical region, the N-peptides are in light grey, while the C-peptideare in dark grey.

[0016]FIG. 6 is the sequence of 6-Helix and 5-Helix. The predictedhelical segments are designated by the stacked sequence.

[0017]FIG. 7 is a ribbon diagram of one of the possible α-helicalarrangements of 5-Helix. The N-helical trimer is light grey, theC-helical regions are in dark grey and the extended loop regions are inblack (based on the structure of D. C. Chan, et al. (Cell 89, 263-273(1997)).

[0018]FIG. 8 shows images of cell-cell fusion assay titrationexperiments. The syncytia (representing fused cells) are blue in theimage while debris is brown.

[0019]FIG. 9 shows images of cell-cell fusion assay competitionexperiments. The amount of syncytia are recorded for cultures incubatedin 200 nM 6-Helix or 5-Helix with increasing amounts of C34 peptide.

[0020]FIG. 10 is a schematic of the design of the Five-Helix constructs.The schematic diagram depicts the linkage pattern of the basic 5-Helixconstruct. Three different C-termini were added. In 6-Helix, aHis-tagged C-peptide is attached to 5-Helix in order to mimic thecomplete six-helix bundle of the trimer-of hairpins. The N40 and C38sequences (alternately joined using short Gly/Ser linkers) are derivedfrom the N- and C-peptide regions of HIV HXB2 gp41. (The red- andblue-boxed residues depict the sequences of the N36 (SEQ ID NO.: 11) andC34 (SEQ ID NO.: 12) peptides, respectively.)

[0021] FIGS. 11A-11C show amino acid sequences of peptides (SEQ ID NOS.:1-10) of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The conformation of a major part of the ectodomain of the gp41molecule consists of a trimer-of-hairpins structure. The core“trimer-of-hairpins” is comprised of a central three-stranded N-helixcoiled coil surrounded by three outer C-helices, forming a bundle with atotal of six helices. The trimer-of-hairpins is a common structuralelement involved in the fusion of many enveloped viruses, suggesting acritical role for this motif in promoting membrane fusion. In HIV gp41,the core of the trimer-of hairpins is a bundle of six α-helices (formedby the C-terminal regions of three gp41 ectodomains) packed in anantiparallel manner against a central, three-stranded coiled coil(formed by the N-terminal regions of the gp41 molecules) (M. Lu et al.,J. Mol. Biol. 290, 1031-1044 (1995); D. C. Chan et al., Cell 89, 263-273(1997); W. Weissenhorn et al., Nature 387, 426-430(1997)); K. Tan etal., Proc. Natl. Acad. Sci. USA 94, 12303-12308 (1997). Because thefusion peptide region, which inserts into the cellular membrane, islocated at the extreme N-terminus of gp41, and the C-terminal region isadjacent to the transmembrane helix anchored in the viral membrane, thetrimer-of-hairpins motif serves to bring the two membranes together.This is illustrated schematically in FIG. 1A. The N-helices (one fromeach subunit of the trimer) form highly conserved hydrophobic groovesinto which the C-helices pack. It is generally agreed that formation ofthis six-helix structure is required for membrane-fusion to occur.

[0023] The importance of trimer-of-hairpins formation for HIV-1 entryled to the hypothesis that the C-terminal region of gp41 might serve asa target for potential membrane-fusion inhibitors. C-peptides have beenshown to inhibit HIV-1 entry into cells, with IC₅₀ values as low as 1 nMin vitro (C. T. Wild et al., Proc. Natl. Acad. Sci. USA 91, 9770-9774(1994); D. C. Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617(1998)). Evidence suggests that C-peptides work in a dominant-negativefashion by binding to the N-peptide region and disruptingtrimer-of-hairpins formation. If the C-terminal region is accessible (atleast transiently) prior to formation of the trimer-of-hairpins, then itis reasonable to expect that agents that bind to this region of gp41N-terminal will prevent membrane fusion. Consistent with this notion,peptides derived from the gp41 N-terminal region (referred to asN-peptides) are modest inhibitors of HIV-1 membrane fusion. Theinhibitory mechanism of N-peptides has not been determined, in partbecause these peptides have a strong tendency to aggregate.

[0024] Applicants reasoned that a single soluble molecule that containsa folded N-helical core and two of the three C-helices of the coretrimer-of-hairpins would be highly stable and would bind a singleC-peptide with high affinity. As described herein, the hypothesis thatthe C-peptide region of gp41 is a target for inhibition of HIV-1 entryhas been tested. Results of the assessment, also described herein, haveshown that Five-Helix, which binds the C-peptide region of gp41, showspotent inhibitory activity against HIV-1 and against HIV-1 variantscontaining a diverse set of envelope proteins. These results point tothe C-peptide region of HIV gp41 as a viable target to inhibit theformation of the trimer-of-hairpins, which is required for membranefusion (and, thus, HIV infection of cells) to occur.

[0025] Described herein are results that show that a protein that bindsto the C-peptide region of gp41 inhibits HIV entry into cells. Suchproteins are inhibitors of HIV and serve as the basis for development ofadditional anti-HIV agents. They might also be used for generating aneutralizing antibody response that targets the N-terminal region of thegp41 ectodomain.

[0026] Five-Helix, as the proteins are designated, takes advantage ofthe binding properties of the N-helix peptide coiled coil whileminimizing the tendency of the N-peptides to aggregate. In oneembodiment of Five-Helix, five of the six helices that make up the coreof the gp41 trimer-of-hairpins structure are connected with (joined by)short peptide linkers. (See FIG. 1A.) In this embodiment, Five-Helixlacks a third C-peptide helix, thus creating a vacancy in order tocreate a high-affinity binding site for the C-terminal region of gp41.In further embodiments of Five-Helix, the three N-peptide helices andmore than two (but less than three complete) C-peptide helices areconnected with short peptide linkers. In these embodiments, the threeN-peptide helices, two complete C-peptide helices and a portion of thethird C-peptide helix are connected with peptide linkers. The portion ofthe third C-helix can be as few as one amino acid residue of the thirdC-helix or any number of additional amino acid residues of the helix upto, but not including, all of the amino acid residues of the helix.Five-Helix protein of the present invention is soluble underphysiological conditions.

[0027] The core of the trimer-of-hairpins, as formed by individual N-and C-peptides, is already quite stable, with a melting temperature of65° C. Applicants have shown that if 5 of the 6 helices are covalentlyjoined to form a 5-Helix protein, the stability of the core is furtherincreased (the stability is greater than the stability of the 6-Helixcore). Under physiological conditions, Five-Helix is folded, soluble,and stable. It has an α-helical content in close agreement with thevalue predicted from the design. (See FIGS. 2A and 2B.) Inaffinity-interaction experiments, Five-Helix interacts strongly andspecifically with epitope-tagged C-peptides. (See FIG. 2C.) Thisinteraction induces a helical conformation in the bound C-peptide asjudged by the difference in circular dichroism before and after mixing.(See FIG. 2D.) These properties are consistent with the intended designof Five-Helix.

[0028] Five-Helix potently inhibits HIV-1 membrane fusion (nanomolarIC₅₀) as measured by viral infectivity and cell-cell fusion assays. (SeeFIGS. 3A and 3B.) In contrast, a control protein, denoted Six-Helix, inwhich the C-peptide binding site is occupied by an attached C-peptide(i.e., all six helices that constitute the gp41 trimer-of-hairpins havebeen linked into a single polypeptide, as described in Example 1), doesnot have appreciable inhibitory activity. (See FIG. 3A and FIGS. 8 and9). Similarly, a Five-Helix variant, denoted Five-Helix(D4), in whichthe C-peptide binding site is disrupted by mutation of four interfaceresidues (V549, L556, Q563 and V570) to Asp, does not block the membranefusion event even at 1 μM. (See Example 3 and FIG. 3A.) These resultssupport the conclusion that C-peptide binding is the key determinant ofantiviral activity in Five-Helix.

[0029] The inhibitory activities of 5-Helix and C-peptides are expectedto be antagonistic: when 5-Helix binds C-peptide, the amino acidresidues thought to be responsible for the antiviral activities of eachinhibitor are buried in the binding interface. Indeed, mixtures of5-Helix and C34 [a well characterized and potent peptide inhibitor withan IC₅₀ of approximately 1 nM] display a dose-dependent antagonisticeffect (FIG. 3B). In the presence of 5-Helix, high-potency inhibition byC34 is only observed when the peptide is in stoichiometric excess (FIG.3B).

[0030] Five-Helix inhibits infection by viruses pseudotyped with avariety of HIV-1 envelope proteins (from clades A, B, and D) withsimilar potency (FIG. 3D). This broad-spectrum inhibition likelyreflects the highly conserved interface between the N- and C-terminalregions within the gp41 trimer-of-hairpins structure (FIG. 4). Theresidues in the C-peptide region of gp41 that are expected to makecontact with 5-Helix are highly conserved in HIV-1, HIV-2, and SIV (FIG.4).

[0031] As a potent, broad-spectrum inhibitor of viral entry, Five-Helixmay serve as the basis for the development of a new class of therapeuticagents against HIV-1. Although they typically require parenteraladministration, protein-based therapeutics can be practical, asexemplified by insulin, growth hormone, tissue plasminogen activator,granulocyte-colony stimulating factor, and erythropoietin.Alternatively, Five-Helix could be expressed endogenously (e.g., viagene therapy) with secretion into the bloodstream. If Five-Helix wereexpressed endogenously in HIV-infected cells, it could inhibitintracellular folding and transport of gp160. Five-Helix, Five-Helix(D4), and Six-Helix are also potential reagents for small-moleculedrug-screening purposes. Five-Helix offers a great deal of flexibilityin the design of variants with better therapeutic characteristics. Inprinciple, Five-Helix can be modified extensively, except at itsC-peptide binding site, to alter its immunogenic, antigenic,bioavailability, or inhibitory properties. For example, the C-peptidebinding site might be lengthened, shortened, or shifted in the gp41sequence in order to optimize inhibitory potency by targeting differentregions of the gp41 ectodomain.

[0032] It would be desirable to generate neutralizing antibodies thatmimic the binding properties of Five-Helix. The broadly neutralizingability of Five-Helix most likely stems from its interaction with thehighly conserved residues in the C-peptide region of gp41 (FIG. 4).Unstructured C-peptide immunogens may not elicit broadly neutralizingantibodies because the linear sequence of the gp41 C-peptide region isvariable among different HIV-1 strains. Such unstructured C-peptides donot have a long region of conserved amino acids residues. Rather,conserved animo acid residues and nonconserved residues areinterspersed. However, constraining C-peptides or C-peptide analoguesinto a helical conformation (e.g., as in the C-peptide region when itbinds Five-Helix) may lead to useful immunogens in the effort to developan AIDS vaccine. FIG. 4 is a helical wheel diagram depicting theinteraction of Five-Helix with the C-peptide region of gp 41. As shown,on the helical wheel, the whole “face” is comprised of conserved oridentical amino acid residues. As also shown, there is a high degree ofconservation in the a and d positions of the C-peptide region of HIVgp41. Peptides from the C-terminal region of the gp41 ectodomainconstrained in such a manner that they present highly conserved aminoacid residues on a single face of the molecule (such as in positions a,d and e in FIG. 4) can be produced. They can be used as immunogens toproduce antibodies that will presumably bind those amino acid residuesin the corresponding unconstrained peptide (C-peptide region of HIV gp41) and, thus, mimic the binding characteristics of Five-Helix. Forexample, antibodies that bind some or all of the highly conserved(identical and/or conserved) amino acid residues in C38 (see FIG. 4) canbe produced. Such antibodies, which mimic the binding of Five-Helix,will work, in effect, as a preventive or vaccine by reducing orpreventing the activity (binding) of Five-Helix. Such antibodies toconstrained peptides from the C-terminal region of HIV gp41 ectodomainare a subject of this invention.

[0033] Intriguingly, the epitope for 2F5, the only known humanmonoclonal antibody directed against gp41 with broad neutralizingactivity, is located immediately C-terminal to the C-peptide regiontargeted by Five-Helix (T. Muster, et al., J. Virol. 67, 6642-6647(1993); M. Purtscher, et al., AIDS 10, 587-593 (1996)). The core of the2F5 epitope (Leu-Asp-Lys-Trp; residues 663-666 in the HIV HXB2 gp160sequence) is highly conserved (81% identity) across the same set ofHIV-1, HIV-2, and SIV isolates used to generate FIG. 4. However, someHIV-1 escape variants to 2F5 neutralization do not contain mutations inthe epitope sequence, suggesting that inhibition by 2F5 may involverecognition of additional determinants. The conformation of the2F5-bound epitope remains unknown, but antibodies elicited withfragments of gp41 containing this sequence do not possess significantvirus-neutralizing activity (T. Muster, et al., J. Virol. 68, 4031-4034(1994); L. Eckhart, et al., J. Gen. Virol. 77, 2001-2008 (1996)). Itremains to be seen if 2F5 inhibits infection by interfering withtrimer-of-hairpins formation.

[0034] Further, Five-Helix itself is a vaccine candidate. Thepossibility of eliciting an antibody response against transientlyexposed conformations of proteins involved in HIV-1 fusion has beensuggested (R. A. LaCasse, et al., Science 283, 357-362 (1999)). Onepossible well-defined target is the N-terminal coiled coil that isexposed in the prehairpin intermediate (D. M. Eckert, et al., Cell 99,103-115 (1999)). A 5-Helix-like intermediate may be exposed during thefusion process, and, in this case, antibodies directed against 5-Helixmay inhibit viral entry.

[0035] Results described herein point to the C-peptide region of HIV-1gp41 as a viable target to inhibit the formation of thetrimer-of-hairpins. Structural and computational methods predict similartrimer-of-hairpins motifs for viruses in many diverse families,including orthomyxoviridae, paramyxoviridae, filoviridae, retroviridae,and others. Moreover, in some of these cases, inhibition of viral entryby peptides analogous to the C-peptides of gp41 has been demonstrated.Thus, the Five-helix design approach may offer a widely applicablestrategy for inhibiting viral infections.

[0036] In addition, Five-Helix provides a means to study a formedC-peptide binding site in detail, which cannot be done with aggregableN-peptides. The exposed C-peptide binding site in this Five-Helixmolecule is useful to identify or design molecules that bind to theN-helical core of gp41 and can be further assessed, using known methods,for their ability to inhibit fusion of the HIV membrane with themembrane of a mammalian cell, such as a human cell, thus inhibiting(reducing or preventing) infection of the cell. Further, Five-Helix canbe assessed for its ability to bind to the C-helical region of gp41 andinhibit its function. The N-helical core of gp41 is highly conserved (interms of amino acid composition) and, thus, it is likely that 5-Helixand variants thereof will be broadly neutralizing against a variety ofclinical HIV strains and, thus, useful therapeutically.

[0037] The Five-Helix protein, which is based upon the known structureof the gp41 ectodomain, consists, in one embodiment, of three N-peptidesand two C-peptides covalently linked and arranged to fold into asubstantial part of the N-helical core with two of the three C-helixbinding sites occupied by C-peptide. The remaining C-peptide bindingsite of the N-peptide is exposed. The site exposes an epitope that is 40amino acids in length. In addition, it is expected that the backboneatoms of the site are rigidly held in a structured conformation, as theN-peptide core is locked into place by the outer two C-peptides.

[0038] In single letter amino acid code, the amino acid sequence of oneembodiment of Five-Helix is the following:MQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARI LAGGR (SEQ IDNO.: 1).

[0039] In single letter amino acid code, the amino acid sequence of6-Helix is the following:MQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGRGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLGGHHHHHH (SEQ ID NO.: 2).

[0040] Five-Helix protein can be produced by a variety of methods. Forexample, it can be produced, as described in Example 1, from a largerprotein, such as 6-Helix, by enzymatic (trypsin) digestion.Alternatively, it can be produced, using known methods and expressionsystems, by expressing Five-Helix protein-encoding DNA, which can be asingle DNA that encodes the entire Five-Helix protein or two or more DNA“units”, each of which encodes a portion (e.g., one or more N helices,one or more C helices) of N-Helix protein. The yield of expression andpurification of Five-Helix can be significantly improved by directexpression of the Five-Helix gene in an appropriate host cell, such asE. coli. In this approach, the Five-Helix gene encodes the residuespresent in the final Five-Helix protein. A C-terminal His-tag can beattached to facilitate purification (with or without a protease cleavagesite to later remove the tag). The protein can then be used directlywithout the proteolytic cleavage and unfolding steps required forproducing Five-Helix starting from Six-Helix. This Five-Helix moleculemay be expressed as a folded active molecule, allowing its use inbiological selections or screens for optimizing its properties.Alternatively, protein synthetic methods can be used to produceFive-Helix protein. The five helices of Five-Helix can be joinedcovalently (such as by means of a linker of at least one (one or more)amino acid residues) or by other means which results in formation of aprotein which is stable under physiological conditions and is correctlyfolded such that the remaining surface of Five Helix is presented sothat it is available to bind C34 peptide. In the embodiments in whichthere are three N-helices and more than two (but less than threecomplete) Chelices, the helices can be similarly joined.

[0041] Five-Helix can have a wide variety of sequences, both in the N-and C-helix regions and in the linker components, and can be comprisedof L-amino acid residues, D-amino acid residues or a combination of bothL- and D-amino acid residues. The amino acids residues can be modified.Five-Helix can include amino acid residues in addition to those of thehelices and linkers (e.g., to stabilize the molecule). It is likely thatthe Five-Helix described here can be altered to enhance stability andactivity. Minor changes in the design of the loops connecting the N- andC-helices (both in length and composition) and the exact borders of theN- and C-helices are likely to have significant effects on thestability, yield, and activity of Five-Helix.

[0042] As currently constructed, Five-Helix exposes a C-peptide bindingsite encompassing 40 amino acids along the N-helical core. A strategyfor exposing shorter segments of the C-peptide binding site on 5-helix(or related molecules) involves attaching a short C-peptide sequenceonto the longer exposed epitope. A molecule of this type might aid inthe development of drugs targeted specifically to a shorter epitopealong the N-helical core. For instance, a single pocket region (similarto that found in IQN17; D. M. Eckert, et al., Cell 99, 103-115 (1999))could be exposed in Five-Helix by binding a C-peptide that lacks theresidues that bind there (the first 10 or so residues of C34). Theseshort C-peptide sequences could be attached to Five-Helix through avariety of means, including covalent crosslinking or merely extendingthe sequence of Five-Helix to cover part of the exposed epitope.

[0043] Five-Helix is useful in a variety of contexts. As describedherein, Five-Helix is a potent inhibitor of viral membrane fusion, and,thus, acts on the virus before it enters the cell (unlike currentpractical therapy) which acts on HIV-infected cells. Five-Helix issoluble and has been shown to be stable under the conditions describedherein. It should also be possible to generate 5-Helix variants with anincreased molecular weight (by oligomerization or tethering to a largeprotein) to reduce the rate of kidney clearance. In addition, Five-Helixdimers can be made by disulfide crosslinking, to produce a moleculefiltered to a lesser extent than the Five-Helix “monomer”. Thus, it isreasonable to expect that dimers might have an enhanced bioavailabilitywhen compared to that of the C-peptides.

[0044] Five-Helix prevents virus from entering cells, unlike standardtherapy that targets viral proteins after viral entry, and thus,Five-Helix can be used prophylactically to prevent infection or reducethe extent to which infection occurs. One use for such a therapeutic isin the event of a needlestick injury, such as might occur in a hospitalor in settings in which needles contaminated with HIV are shared. Forexample, an individual who is stuck with a needle and is or might beinfected with HIV can receive a sufficient quantity of Five-Helix(therapeutically effective quantity) in one or more dose(s) in order toprevent or reduce HIV entry into cells. Five-Helix can be administered,for example, by intravenous or intramuscular injection.

[0045] In one embodiment of the present invention, Five-Helix is used toreduce HIV infection in an individual. In this embodiment, Five-Helix isadministered, either as Five-Helix itself or via expression ofFive-Helix-encoding DNA in appropriate host cells or vectors, to anindividual in sufficient quantity to reduce (totally or partially) HIVinfection of the individual's cells. That is, a dose of Five-Helixsufficient to reduce HIV infection (an effective dose) is administeredin such a manner (e.g., by injection, topical administration,intravenous route) that it inhibits (totally or partially) HIV entryinto cells. In one embodiment, a gene therapy approach is used toprovide the effective dose, by introducing cells that express Five-Helixprotein into an individual. Five-Helix can be administered to anindividual who is HIV infected to reduce further infection, or to anuninfected individual to prevent infection or reduce the extent to whichinfection occurs.

[0046] Pharmaceutical compositions which comprise Five-Helix in anappropriate carrier (e.g., a physiologically acceptable buffer) are asubject of this invention. They are useful for preventive andtherapeutic purposes and can be administered via a variety of routes(e.g., injection, topical administration, intravenous route).

[0047] Five-Helix appears to present a single, intact C-helix bindingsite and, thus, is useful for screening for drugs that inhibit membranefusion. Five-Helix exposes a larger, more rigid target for potentialdrug screens than does IQN17. The molecules 6-Helix and 5-Helix(D4) area useful negative control in these studies.

[0048] The Five-Helix exposed epitope can also be used as an antigen forproducing antibodies, particularly neutralizing antibodies using knownmethods. The antibodies can be monoclonal or polyclonal.

[0049] The serum stability of Five-Helix can be tested, using knownmethods, to ascertain its therapeutic potential. If Five-Helix isdegraded, the most likely point of attack/degradation is theglycine/serine linker regions. In this case, different linker regionscan be generated and tested (see below). The inhibitory ability of theseanti-Five-Helix sera and ascites can be tested using standard fusionassays.

[0050] The outside surface of Five-Helix can be varied, for example, toenhance bioavailability, decrease toxicity and avoid immune clearance.Since Five-Helix exhibits potent inhibitory activity, whereas the6-Helix bundle does not, it is the exposed groove, including the pocketregion, that is responsible for inhibition. The rest of the moleculesimply provides a scaffold for displaying the exposed groove. Therefore,this scaffold can be modified without adversely affecting the inhibitoryactivity of Five-Helix. Modification of the scaffold may provide severaladvantages. First, it would facilitate procedures in which multipleadministrations of Five-Helix are required. For example, when Five-Helixis used as an anti-HIV therapeutic agent, multiple doses might berequired. After extended administration, individuals might developantibodies to Five-Helix that are likely to increase its clearance fromthe body. The availability of multiple versions of 5-Helix would help tocircumvent this problem by evading pre-existing antibodies. Second, itmay be possible to design versions of Five-Helix, for example byintroducing glycosylation sites on the external surface, in which thescaffold is less immunogenic. For vaccine studies, this modificationwould help to bias the immune response toward the exposed groove asopposed to the scaffold.

[0051] The observation that binding the gp41 C-helical region preventsHIV infection suggests a strategy for constructing an HIV vaccine.Analogous to inhibition of HIV by C-peptides, Five-Helix likely inhibitsgp41 by binding to a fusion intermediate of gp41 called the prehairpinintermediate. Whereas the C-peptide inhibitors function by binding tothe N-peptide region of this intermediate, Five-Helix likely functionsby binding to the C-peptide region. These considerations stronglysuggest that the C-peptide region of gp41 is a good drug target for thedevelopment of HIV entry inhibitors. Moreover, it may be possible to useC-peptide-based constructs as immunogens to elicit neutralizingantibodies. In the case of Five-Helix, the target of inhibition is ahelical conformation of the C-peptide region, but reagents targetingother conformations of the C-peptide region may also have inhibitoryactivity.

[0052] Recent vaccine studies (R. A. LaCasse, et al., Science 283,357-362 (1999)) suggest that intermediates of the envelope-mediatedfusion process can elicit strongly neutralizing antibodies. Antibodiesto such fusion intermediates would target conserved regions of theenvelope proteins and therefore would be likely to neutralize a broadrange of viral strains. Antibodies to the C-peptide region would targeta region that is highly conserved and critical to the fusion process.

[0053] The trimer-of-hairpins is a common feature of many viral membranefusion proteins. It has been observed in crystal structures ofInfluenza, Ebola, SV5 (simian parainfluenza virus 5), and RSV (humanrespiratory syncitial virus). In addition, many other members of theretrovirus, paramyxovirus, and filovirus families are predicted tocontain this motif. A similar structure has been observed in theassociated vertebrate vesicle fusion proteins. The basic strategydescribed herein can be applied to any of these systems in order toinhibit fusion. One subject of this invention is a method of inhibitingformation of the trimer-of-hairpins of an enveloped virus (a virus thatcomprises a viral envelope protein) by contacting the virus with a drugthat binds a viral envelope protein (e.g., the C peptide region of aviral envelope protein) and inhibits formation of the trimer-of-hairpinsof the enveloped protein.

[0054] The present invention is illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLE 1 Production of 5-Helix

[0055] The design of 5-Helix was based on the N36/C34 six-helix bundlecrystal structure (D. C. Chan, et al., Cell 89, 263-273 (199,7)). Forthe 5-Helix protein, each peptide region was extended (compared with N36and C34) by three residues on its N-terminus and one residue on itsC-terminus, generating the final N40 and C38 segments (representingresidues 543-582 and 625-662 of HIV-1 HXB2 gp160, respectively). ThreeN40 and two C38 segments were joined using a -GGSGG-linker after N40 anda -GSSGG-linker after C38. All constructs include an N-terminal Met fortranslation initiation. Two distinct 5-Helix proteins that differ onlyat their C-termini were generated for this study: (i) His-tagged5-Helix, which ends in -GG(H)6, and (ii) untagged 5-Helix, which ends in-GGR. In addition, a third construct, denoted 6-Helix, was generated inwhich the 5-Helix backbone was connected to the His-tagged C-peptide,C37-H6 (see Example 4), through a trypsin-cleavable linker (-GGR-) (seeFIGS. 10 and 11A-1C).

[0056] All DNA constructs were assembled from PCR cassettes sequentiallycloned into the pAED4 vector [D. S. Doering, P. Matsudaira, Biochemistry35, 12677-12685 (1996)] using E. coli XL1-Blue (recA—strain,Stratagene). All proteins were recombinantly expressed in E. coli strainRP3098 grown in 2×YT to an OD (590 nm) between 0.5-0.7 before inductionwith IPTG (0.4 mM) for 3 hours. Bacterial pellets were resuspended inTris/NaCl buffers (Qiaexpressionist booklet, March 1999, Qiagen)supplemented with Complete EDTA-free protease inhibitor tablets (Roche),and subsequently frozen at −20° C. until the day of purification. Thawedresuspensions were lysed (sonication or French press) and centrifuged(35,000×g for 30 minutes) to separate the soluble fraction frominclusion bodies.

[0057] His-tagged 5-Helix (generated from plasmid p-5HelixH6) waspurified directly from the inclusion bodies resuspended in 8 M urea inTBS (50 mM Tris, pH 8.0, 100 mM NaCl) and 10 mM imidazole. The mixturewas clarified by centrifugation (35,000×g for 30 minutes) before bindingto a Ni-NTA agarose (Qiagen) column at room temperature. Protein waseluted in 6 M urea/TBS/100 mM imidazole in 40 ml (˜5 column volumes).The protein was refolded by slow dripping into a one liter, stirredsolution of 20 mM Tris (pH 8.0) at room temperature. Refolded proteinwas then reconcentrated by passage over a Ni-NTA agarose column andeluted with 20 ml (˜2 column volumes) of 100 mM imidazole in TBS.

[0058] Untagged 5-Helix was produced via proteolysis of 6-Helix (seebelow) to generate a 5-Helix/C37-H6 complex. Following digestion withtrypsin (1:200 weight ratio in TBS at room temperature for 1 hour,Sigma), the 5-Helix/C37-H6 complex was bound to Ni-NTA agarose andwashed extensively to remove excess trypsin. The beads were resuspendedin 8 M GuHCI/TBS and heated (70° C.) in order to denature the complex.The nonbinding fraction, containing denatured 5-Helix, was sequentiallyIdialyzed into 8 M urea/20 mM Tris, pH 8.0 (4 hours at room temperature)and 4 M urea/20 mM Tris, pH 8.0 (overnight at 4° C.). The protein wasloaded onto a DEAE column (Fastflow, Pharmacia) and a reverse ureagradient (4 M to 0 M urea in 20 mM Tris, pH 8.0) was run over 20 columnvolumes in 4 hours at room temperature. The protein was eluted from theDEAE resin using a NaCl gradient (0 to 300 mM) in 20 mM Tris, pH 8.0 (10column volumes). 6-Helix (generated from plasmid p-6-Helix) was purifieddirectly from the soluble fraction of the bacterial lysate. The solutionwas passed over Ni-NTA agarose column and eluted with an imidazolegradient (10-250 mM) in TBS over 10 column volumes.

[0059] For all proteins, monomers were separated from aggregates by gelfiltration (Sephacryl S200 HR or Superdex 75) in TBS. The proteinswere >95% pure as judged by SDS-PAGE and can be concentrated to at least3 mg/ml. The concentrations of all peptides and proteins were determinedby absorbance at 280 nm in 6 M GuHCl [H. Edelhoch, Biochemistry 6,1948-1954 (1967)].

EXAMPLE 2 Assessment of the Specificity of 5-Helix/C-peptide Interactionand of Inhibition by 5-Helix of Membrane Fusion

[0060] The specificity of 5-Helix/C-peptide interaction has been testedusing a His-tagged C-peptide (C37-H6, independently expressed in E. coliand purified through reverse-phase HPLC) and Ni-agarose precipitation.In TBS with 30 μM of C37-H6, 16 μM of 5-Helix is completely precipitatedby Ni-agarose. Addition of 150 μM C34 (no His-tag, chemicallysynthesized and purified over HPLC) substantially reduces the amount ofprecipitated 5-Helix. The effective competition of C37-H6 and C34indicates that 5-Helix binds C-peptide in a specific manner. The CDexperiments and competitive binding assays suggest that 5-Helix foldsinto the predicted conformation. That is, the results support theprediction that 5-Helix contains an exposed C-peptide binding site.

[0061] Assays were carried out to assess the ability of 5-Helix tointeract with the Cregion of gp41 and inhibit function of the fusionprotein. This inhibition of membrane fusion by 5-Helix and 6-Helix wasassessed using a cell-based assay. Proteins 5-Helix and 6-Helix areserially diluted in modified DMEM media with 5% FCS and aliquoted intoslide chambers. HELA cells (4×10⁴) expressing CD4 and coreceptors andcontaining a β-galactosidase gene under the control of the Tat promoterare added. CHO cells (2×10⁴) expressing gp160 (precursor protein togp120/gp41 ) and Tat are also added. The 400 μl miniculture is incubatedat 37° C. for 8 to 24 hours; fused cells (syncytia) will transcribe andtranslate β-galactosidase. The cells are fixed in gluteraldehyde andexposed to X-gal/Fe solution for one hour. Syncytia that containβ-galactosidase turn blue-green. In this assay, 5-Helix demonstrates apotent inhibition of syncytia formation, with an IC₅₀ of 10-20 nM; inone assay the IC₅₀ was 13 nM. 6-Helix does not block fusion appreciablyeven at 1 μM concentrations.

[0062] In order to verify the specificity of the 5-Helix exposed epitopeas the inhibitory agent and to rule out a contaminant, mixingexperiments with C-peptide have been performed. 5-Helix, at 200 nMconcentration, is mixed with C34 at 100, 166, 190 and 210 nM. At theconcentrations used, free 5-Helix and free C34 should inhibit almost allof the syncytia in the miniculture. In 5-Helix/C34 mixes where C34 is inexcess of the 5-Helix (i.e., at 210 nM) syncytia formation is notblocked in the 5-Helix/C34 mixes where C34 concentration is less thanthat of 5-Helix. By contrast, C34 in the presence of 6-Helix blocks allsyncytia formation.

[0063] The inhibitory potentials of 5-Helix and 6-Helix have beenreproduced in viral fusion experiments. HIV, modified to contain aluciferase reporter gene, is mixed with human osteosarcoma (HOS) cellsexpressing CD4 and coreceptor in the presence of diluted protein for 6hours at 37° C. The virus solution is replaced, and the HOS culture isincubated 48 hours more in fresh media. Luciferase activity is measuredin a luminometer. In this assay, 5-Helix inhibits luciferase activitywith an IC₅₀ less than 10 μM. Again, 6-Helix shows no appreciable blockup) to 1 μM (FIGS. 3A and 3C).

EXAMPLE 3 Design and Assesment of 5-Helix (D4)

[0064] In 5-Helix(D4), four highly conserved residues in the C-peptidebinding site of His-tagged 5-Helix (Val549, Leu556, Gln563, and Val570)were mutated to Asp in the final (third) N40 segment. The construct[p-5-Helix(D4)] was recombinantly expressed and purified in the samemanner as the His-tagged 5-Helix. The His-tagged 5-Helix and 5-Helix(D4)proteins have the same ellipticity: for both, [θ]₂₂₂=−28,100±1500 degcm² dmol⁻¹ (˜100% of the predicted helical content) at 4° C. in TBS, andboth proteins are extremely stable to thermal denaturation (Tm>98° C.)in TBS, as well as to GuHCl chemical denaturation (Cm values 6 M for5-Helix(D4); ˜7.2 M for the His-tagged 5-Helix) at 25° C. The slightlydecreased stability of 5-Helix(D4) likely reflects the low helicalpropensity and charge of the Asp residues which, in this context, areplaced within a predominantly hydrophobic groove on the surface of5-Helix.

EXAMPLE 4 His-tagged C-peptide C37-H6

[0065] Peptide C37-H6 is a His-tagged C-peptide of the followingsequence: GGHTTWMEWDRENNYTSLIHSLIEESQNQQEKNEQELLGHHHHHH (SEQ ID NO.: 5).The peptide is derived from HIV-1 HXB2 residues 625-661 (underlined) andcontains the entire C34 sequence (W628 to L661). C37-H6 is produced fromthe tryptic digestion of a recombinantly expressed construct, p4-NC1.1,consisting of one N40 segment joined to C37-H6 through a -GGR-linker.Following expression, NC1.1 is purified from the soluble fraction ofbacterial lysates in the same manner as 6-Helix. Trypsin digestion (sameconditions as for untagged 5-Helix) generates C37-H6, which is thenpurified to homogeneity by reverse phase HPLC using a Vydac C-18 columnand a linear gradient of acetonitrile in water containing 0.1%trifluoroacetic acid. The identity of C37-H6 was confirmed by massspectrometry (MALDI-TOF, PerSeptive). Like C34, C37-H6 is a potentinhibitor of HIV-1 membrane fusion, with an IC₅₀=1 nM in the cell-cellfusion assay.

[0066] The data in FIGS. 2A-2D were generated using the untagged versionof 5-Helix, but similar results were obtained with the His-taggedversion [see Example 3]. The CD (Aviv 62 DS) experiments were performedin TBS buffer unless otherwise stated. In FIG. 2B, the proteinconcentration was 1 mM for the TBS sample and 0.54 mM for the GuHCI/TBSsample. In FIG. 2D, a quartz mixing cell (Helma) with 1 ml chambers(4.375 mm/chamber pathlength) was utilized. The polypeptides were at aconcentration of 5.9 mM (5-Helix) and 6mM (C37-H6) in 20 mM Tris, pH8.0/250 mM NaCl before mixing.

[0067] The 5-Helix precipitation experiment (FIG. 2C) was performed in20 ml TBS with 16 mM untagged 5 Helix, 30 mM His-tagged C37-H6, and/or150 mM C34. The solution was added to 10 ml of Ni-NTA agarose andincubated at room temperature for 10 minutes. After the unboundsupernatant was removed, the beads were washed twice with 1 ml TBS andthen eluted with 500 mM imidazole. The Ni-bound and unbound samples wererun on a 16.5% Tris-Tricine polyacrylamide gel (Biorad) and stained withGel-code Blue (Pierce).

EXAMPLE 5 His-tagged 5-Helix

[0068] All data in FIGS. 3A-3C were generated using His-tagged 5-Helix(see Example 1). The cell-cell fusion assays (FIG. 3B) were performed asdescribed (D. C. Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617(1998)). Inhibition of viral infectivity was studied using a recombinantluciferase reporter assay slightly modified from that previouslydetailed (D. C. Chan, et al., Proc. Natl. Acad. Sci., USA, 95,15613-15617 (1998)). Briefly, pseudotyped viruses were generated from293T cells cotransfected with an envelope-deficient HIV-1 genomeNL43LucR⁻E⁻[B. K. Chen, et al., J. Virol. 68, 654-660 (1994)] and one offour gp160 expression vectors: pCMV-HXB2 (D. C. Chan, et al., Proc.Natl. Acad. Sci., USA, 95, 15613-15617 (1998), pEBB-JRFL (kindlyprovided by B. K. Chen), pSVIII-UG024.2, and pSVIII-RW020.5. Theplasmids pSVIII-UG024.2 and pSVIII-RW020.5 were obtained from the NIHAIDS Reagent Program (F. Gao, B. Hahn, and the DAIDS, NIAID) and codefor envelope protein from primary HIV-1 isolates. Supernatantscontaining virus were prepared as previously described (D. C. Chan etal., Proc. Natl. Acad. Sci. USA 95, 15613-15617 (1998)) and used toinfect either HOS-CD4 cells (HXB2 and UG024.2) or HOS-CD4-CCR5 cells(JRFL and RW020.5). Cells were obtained from the NIH AIDS ReagentProgram (N. Landau). In FIG. 3A, viral infectivity assays were performedin the standard 24-well format (D. C. Chan et al., Proc. Natl. Acad.Sci. USA 95, 15613-15617 (1998)). The data in FIG. 3C were obtained fromassays conducted in 96-well format: virus-containing supernatant (10 ml)and media (90 ml) were overlaid onto HOS cells at 50% confluency.Following two days of incubation at 37° C., the cells were harvested in100 ml lysis buffer (Luciferase Assay System, Promega), of which 10 mlwas analyzed per manufacturer's protocol. The IC₅₀ values werecalculated by fitting the 5-Helix titration data to a Langmuir function[normalized hiciferase activity=1/(1+[5-Helix]/IC₅₀)].

[0069] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. Five-Helix protein which is soluble underphysiological conditions and comprises the three-N-helices and at leasttwo, but not three complete, C-helices of the trimer of hairpinstructure of HIV gp41, wherein the component helices are separated bylinkers.
 2. Five-Helix of claim 1 wherein the linker comprises at leastone amino acid residue linker.
 3. Five-Helix protein of claim 2 whereinthe protein binds the C-peptide region of HIV gp41.
 4. Five-Helixprotein of claim 2 which includes the three N-helices and two C-helicesof the trimer of hairpin structure of HIV gp41.
 5. Five-Helix comprisingSEQ ID No.:
 1. 6. Six-Helix comprising the three N-helices and the threeC-helices of the trimer hairpin structure of HIV gp41, wherein thecomponent helices are separated by linkers.
 7. Six-Helix proteincomprising SEQ ID NO.:
 2. 8. A method of identifying a compound ormolecule that binds Five-Helix and inhibits HIV infection of mammaliancells, wherein the compound or molecule to be assessed is referred to asa candidate inhibitor, comprising combining a candidate inhibitor andFive-Helix, under conditions appropriate for binding of an inhibitor andFive-Helix to occur and determining if binding occurs, wherein ifbinding occurs, the candidate inhibitor is a compound or molecule thatbinds Five-Helix.
 9. The method of claim 8 further comprisingdetermining if the compound or molecule that binds Five-Helix inhibitsHIV infection of mammalian cells in a cell-based assay.
 10. A method ofeliciting an immune response to HIV in an individual, comprisingintroducing, by an appropriate route, a composition comprising 5-Helixand a physiologically acceptable carrier, in a dose sufficient to elicitthe immune response in the individual.
 11. Five-Helix that binds theC-peptide region of HIV gp41 and inhibits HIV infection of mammaliancells.
 12. Five-Helix of claim 9 that inhibits HIV infection of humancells.
 13. Five-Helix that interferes with formation of the HIV gp41trimer of hairpin structure and inhibits HIV infection of cells. 14.Five-Helix that inhibits fusion of HIV and mammalian cell membranes, asmeasured by viral infectivity assay, cell-cell fusion assay or both. 15.Five-Helix of claim 14 , wherein the mammalian cell membranes are humancell membranes.
 16. Five-Helix of claim 14 wherein the protein inhibitsHIV membrane fusion at nanomolar IC₅₀, as measured by viral infectivityassay or cell-cell fusion assay.
 17. A method of eliciting aneutralizing anti-HIV response in an individual, comprisingadministering to the individual Five-Helix that binds the C-peptideregion of HIV gp41.
 18. A method of inhibiting HIV infection of cells inan individual, comprising administering to the individual Five-Helix insufficient quantity and by an appropriate route for Five-Helix to bindthe C-terminal region of HIV gp41, whereby HIV membrane fusion and HIVinfection of cells are inhibited.
 19. A method of inhibiting fusion ofHIV and human cell membranes in an individual, comprising administeringto the individual a drug that inhibits formation of thetrimer-of-hairpins of HIV gp41, thereby inhibiting fusion of HIV andhuman cell membranes.
 20. The method of claim 19 wherein the drug isFive-Helix or a neutralizing antibody that mimics the binding propertiesof Five-Helix.
 21. A method of inhibiting formation of thetrimer-of-hairpins of an enveloped virus, comprising contacting thevirus with a drug that binds a viral envelope protein and inhibitsformation of the trimer-of-hairpins of the enveloped virus.
 22. Themethod of claim 21 wherein the drug is Five-Helix or an antibody thatmimics the binding properties of Five-Helix.
 23. Five-Helix complex,wherein the complex comprises Five-Helix linked to a molecule that bindsHIV envelope protein.
 24. Five-Helix complex of claim 23 , wherein themolecule that binds HIV envelope protein binds HIV gp120.
 25. Five-Helixcomplex of claim 24 wherein the molecule that binds HIV gp120 is sCD4 oran antibody.
 26. A method of inhibiting fusion of HIV and human cellmembranes in an individual, comprising administering to the individualFive-Helix complex in sufficient quantity and by an appropriate routefor Five-Helix complex to bind HIV envelope protein, wherein Five-Helixcomplex comprises Five-Helix linked to a molecule that binds HIVenvelope protein.
 27. The method of claim 26 , wherein the envelopeprotein is HIV gp120.
 28. An isolated protein selected from the groupconsisting of: (a) (SEQ ID NO.: 1) (b) (SEQ ID NO.: 2) (c) (SEQ ID NO.:3) (d) (SEQ ID NO.: 4) (e) (SEQ ID NO.: 5) (f) (SEQ ID NO.: 6) (g) (SEQID NO.: 7) (h) (SEQ ID NO.: 8) (i) (SEQ ID NO.: 9) (j) (SEQ ID NO.: 10)(k) (SEQ ID NO.: 11) (l) (SEQ ID NO.: 12).